Early and accurate diagnosis of hepatocellular carcinoma (HCC) remains a major clinical challenge, particularly in patients with chronic hepatitis C virus (HCV) infection, where standard biomarkers such as alpha-fetoprotein (AFP) often lack sensitivity and specificity. Numerous biomarkers have emerged from untargeted metabolomics and lipidomics approaches; nevertheless, most of these studies provide only relative amounts and do not perform quantitative analysis as further validation. To address this challenge, we developed and validated a targeted method to quantitatively measure panels of biomarkers previously identified as predictive of HCC in untargeted analyses. A high-throughput (6 min) HILIC-HRMS method, based on a multiplexing-HRMS strategy, was developed and validated to measure a panel of lysophosphatidylcholines (LPCs) and carnitines (CARs) in plasma samples from 92 HCC patients. The method showed satisfactory performances in terms of sensitivity (LODavg: 0.0558 ng/mL, LOQavg: 0.667 ng/mL), accuracy (98.75

The E6 oncoprotein of high-risk human papillomaviruses (HPVs) promotes cervical carcinogenesis by forming a complex with the ubiquitin ligase E6AP to mediate p53 proteasomal degradation. A 15-amino-acid peptide (pep11) was shown to disrupt the HPV16 E6/E6AP complex, to restore p53 stability and to induce apoptosis in HPV16-positive cancer cells. In this study, a short cell-penetrating peptide (CPP) was conjugated to the pep11 N-terminus to improve its solubility and intracellular delivery in HPV-positive cancer cells. CPP-pep11 was synthesized using Boc chemistry. The interactions of CPP-pep11 with HPV16 and HPV18 E6 were modelled using AlphaFold2, while peptide structures were characterised by NMR spectroscopy. HPV16-positive SiHa and HPV18-positive C4-I cervical cancer cells were treated with CPP-pep11 (0.5–20 µM) for 24–72 h. Effects on proliferation and clonogenicity were assessed using xCELLigence and colony formation assays. Peptide uptake and localization were analysed by confocal microscopy and differential cell fractionation, while p53 expression and viability were evaluated by Western blotting and cytotoxicity assays. AlphaFold2 modelling suggested that CPP-pep11 can interact with both HPV16 E6 and HPV18 E6, with a more stable predicted binding to HPV16 E6. CPP-pep11 was soluble in water at 20 µM and, according to NMR analysis, predominantly adopted a disordered conformation. CPP-pep11 efficiently entered the SiHa and C4-I cell membranes, mainly localised to the cytoplasm at lower concentrations (0.5–5 µM) and to the nucleus at higher concentrations (10–20 µM). CPP-pep11 induced dose- and time-dependent inhibition of proliferation, with a significant reduction in colony formation in both cell lines. Treatment also restored p53 levels at 48 and 72 h, consistent with disruption of E6-mediated degradation. In conclusion, CPP-pep11 is a novel peptide-based inhibitor of HPV16 E6 and HPV18 E6 with therapeutic potential in HPV-associated malignancies by restoring p53 levels and inhibiting cancer cell proliferation.

Head and neck squamous cell carcinoma (HNSCC) are a heterogeneous group of tumors linked to diverse risk factors, such as tobacco and alcohol use, as well as human papillomavirus (HPV) infection. HPV16 E6 and E7 oncoproteins are the main players of cell transformation, with the E6*I isoform increasing during neoplastic progression. The aim of this study was to evaluate the role of cellular splicing factors in the production of E6*I in HPV16-related HNSCC. We have evaluated the levels of splicing factors mRNA (HNRNPA1, HNRNPA2B1, SRSF1, SRSF2, SRSF3, BRM and SAM68) as well as HPV16 E6 and E6*I mRNAs by qPCR in HNSCC as well as in SCC152 and SCC154 cell lines. Overall, 42.4% of HNSCC tested positive for HPV16 DNA, and among these 54% expressed E6*I mRNA. The SRSF3, BRM and SAM68 transcripts were significantly higher in HPV-positive compared to HPV-negative HNSCC (p < 0.05), and SAM68 with HPV16 E6*I transcripts concordantly high in both HNSCC and cell lines (r = 0.7). Transduction of LXSN_E6 in HPV-negative PCA5 cell line induced production of E6*I mRNA and overexpression of Sam68 protein. In addition, silencing of SAM68 in SCC152 caused decrease of E6*I RNA and reduced cell growth at 48 hr after siRNA transfection. Higher expression of splicing factors in association with HPV was also confirmed in HNSCC TCGA dataset. In conclusion, our results suggest an interplay between the splicing machinery and HPV16 E6*I in HNSCC. These new observations are crucial for the development of novel therapeutic strategies based on SAM68 inhibitors.

BackgroundMutations in the TP53 gene and telomerase reverse transcriptase promoter (TERTp) are among the most frequent genetic alterations in bladder cancer, but the clinical impact of their co-occurrence has not been fully explored. In this study, we assessed the mutational landscape as well as the prognostic significance of concurrent TERTp and TP53 mutations in a cohort of bladder urothelial carcinoma patients.MethodsUsing data from the cBioPortal database, we retrospectively analysed primary bladder urothelial carcinoma cases profiled with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assays. We investigated the relationships between tumour mutational burden (TMB), microsatellite instability (MSI), and somatic mutations. The Kaplan-Meier method was used to calculate patient overall survival. Log-rank testing and multivariable Cox proportional hazards modelling were used to evaluate prognostic factors.ResultsAmong the 1,111 cancer cases, 416 exhibited concurrent mutations in both TERTp and TP53, 387 harboured mutations exclusively in TERTp, 132 showed mutations only in the TP53 gene, and 176 cases were double wild-type for both genetic regions (wt/wt). Overall survival was significantly longer in the wt/wt group compared to TERTp (HR 1.83, 95% CI 1.27 - 2.62, P<0.001), to TP53 mutant alone (HR 1.84, 95% CI 1.19 - 2.85, P = 0.006) and to TERTp/TP53 (HR 2.32, 95% CI 1.63 – 3.31, P<0.001) mutant groups. The presence of TERTp and TP53 mutations was associated with higher tumour mutational burden (TMB ≥10 mutations/Mb) and increased microsatellite instability (MSI) scores (P < 0.001). The significant association between TERTp and TP53 mutations was independently validated in a separate cohort.ConclusionsBladder urothelial cancer can be stratified into biologically and clinically distinct subtypes on the basis of cancer driver mutations, with concomitant TERTp/TP53 nucleotide changes strongly linked to reduced patients’ overall survival. These results suggest a potential cooperative interaction between mutant TERTp and TP53 in the pathogenesis of bladder cancer, highlighting their significance as prognostic biomarkers and promising targets for novel therapeutic strategies.

The clinical translation of automated HPV detection in Whole Slide Images (WSIs) is challenged by staining variability, sparse viral effects, and the biological continuum between cytology and histology. This work presents a fully automated pipeline for binary patch-level classification of HPV-induced lesions on H&E-stained tissue. The core contribution is a domain-adaptive transfer learning strategy: a ResNet50 backbone is pretrained on the SIPaKMeD cervical cytology dataset rather than ImageNet, then fine-tuned on a target histological cohort. Preprocessing includes adaptive tissue segmentation, blur rejection, and Macenko stain normalization to ensure vendor-agnostic inputs. Evaluated using a strict Leave-One-Patient-Out cross-validation on 42 diagnostic specimens, the SIPaKMeD-based initialization significantly outperforms the ImageNet baseline. This approach achieves higher AUC-ROC scores and superior stability across folds, demonstrating that domain-specific pretraining effectively mitigates data scarcity and class imbalance in digital cervical cancer screening. Under a complementary 5-fold patient-level cross-validation covering all 19 patients of the cohort (133,704 patches, 7181 HPV-positive, prevalence 5.37%), the SIPaKMeD-pretrained model attains a mean test AUC-ROC of 0.694 with a 95% patient-aware bootstrap confidence interval of [0.681, 0.705], consistently above the ImageNet baseline mean of 0.656 obtained on the controlled three-fold ablation.

Cervical cancer is one of the leading causes of tumor-related deaths among women. Chemotherapy in cervical cancer is mainly based on cisplatin, but this drug has limited efficacy; therefore, alternative treatment options are needed. Ferroptosis represents a novel form of cell death. In cervical epithelium, ferroptosis occurs in the early neoplastic stages of papillomavirus infection but shifts to evasion in carcinoma. Combination therapy has the potential to enhance cancer cell death and overcome resistance development. Herein we demonstrate that dimethyl fumarate (DMF), a Food and Drug Administration (FDA)-approved anti-inflammatory drug, induces ferroptosis in cervical cancer cells in a dose-dependent manner and inhibits growth in spheroid models. Cotreatment with DMF and cisplatin significantly decreases cell viability compared to either drug alone. Under DMF/cisplatin combination, cervical cancer cells underwent to glutathione depletion and p53 (re)activation, leading to cell death by both ferroptosis and apoptosis. We found a p53-mediated downregulation of the Solute Carrier Family 7 Member 11 (SLC7A11)/Cystine/Glutamate Transporter (xCT) expression and glutathione levels. Our results suggest that combined administration of DMF and cisplatin, by targeting the dependency of cervical cancer cells on glutathione and (re)activating p53, represents a promising anticancer therapeutic strategy.

Ferroptosis is a type of cell death executed by phospholipid peroxidation in an iron-dependent manner. Ferroptosis plays a central role in inhibiting tumor growth, enhancing the immune response, and is now considered a strategy to combat resistance to anticancer therapies. The oncosuppressor p53 is one of the major regulators of ferroptosis and can either promote or inhibit ferroptosis, depending on the context and/or extent of the damage. p53 governs the transcription of many genes that modulate cell susceptibility to ferroptosis, using this manner of death to fulfill its role as tumor suppressor. The diverse functions of p53 are related to non-coding RNAs (ncRNAs), especially microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), since they can either regulate p53 or be regulated by p53. Therefore, an intricate metabolic network between ncRNAs and p53 ensures the correct response. In this review, we will discuss recent studies on the molecular interplay between p53-mediated ferroptosis and ncRNAs and how this contributes directly or indirectly to the outcome of ferroptosis.

Hepatocellular carcinoma (HCC) is a rapidly progressing disease, frequently caused by hepatitis C virus (HCV) infection and a higher prevalence in males than females. Over 60

Oral HPV DNA and circulating tumor (ct) HPV DNA in plasma were evaluated as potential biomarkers for HPV-associated head and neck cancer (HNC). Samples from HNC patients (n = 132), including 23 oropharyngeal cancers (OPC), and non-HNC controls (n = 10) were analyzed. HPV status was determined using a multiplex bead-based test (E7-MPG) applied to formalin-fixed paraffin-embedded (FFPE) tissues (n = 90), plasma (n = 141), gargle samples (n = 141), and oral swabs (n = 142). HPV DNA was detected in 25.8% of HNC tissues, 12% of plasma samples, 20.6% of gargles and 7% of oral swabs with HPV16 as the most prevalent genotype. Among OPC cases, HPV16 DNA was found in 71.4% of FFPE samples. High concordance was observed between paired OPC tissues and plasma (91.3%) or gargles (95.2%), with moderate concordance for oral swabs (59.1%). Gargle samples alone demonstrated a 100% detection rate for HPV16-positive OPC, regardless of the cT stage, outperforming plasma (86.7%). Combined oral gargle and plasma analyses detected all HPV-positive OPC cases (7/7) at the early cT1 stage. These findings highlight the limited involvement of HPV in non-oropharyngeal HNC compared to OPC, and support gargle and plasma samples as minimally invasive diagnostic tools for detecting HPV-associated OPC.

Background:Head and neck squamous cell carcinomas (HNSCC) have high recurrence and poor prognosis, largely due to delayed diagnosis. Identification of somatic mutations and human papillomavirus (HPV) sequences in tumor DNA shed in the oral cavity may provide non-invasive biomarkers for early HNSCC detection. Objectives:The study aimed to evaluate TERT promoter (TERTp) mutations in tumor DNA extracted from oral rinses as potential biomarkers for head and neck cancers. Methods:TERTp mutations (C228T and C250T) were examined in DNA extracted from oral rinses of 132 HNSCC patients, of whom 63 had paired tumor tissue available for analysis, and from four head and neck squamous cell carcinoma derived cells lines (CAL27, SCC152, SCC154, FaDu) by using droplet digital PCR (ddPCR). TERT gene expression was analyzed in all cell lines by real time PCR. Associations with tumor site, smoking status, and sex were evaluated, and mutant allele frequencies (MAF) quantified. Results:TERTp mutations were identified in 25% of oral rinses (33 out of 132, 95%CI 22.7 - 46.3) and in 27% of tumor tissues (17 out of 63, 95%CI 9.9 - 27.2). Mutation rates were highest in oral SCC (OSCC), present in 50% of oral rinses (n=25/50, 95%CI 16.2 - 36.9) and 46% of matched tumor tissues (n=13/28, 95%CI 6.9 - 22.2), with 96% concordance (kappa value 0.86, 95%CI 67-100). MAF were higher in tumor tissues and correlated with levels in corresponding oral fluids. Mutations were uncommon in non-OSCC cases, being detected in 9.7% of oral rinses and 11% of tumor tissues. In OSCC, TERTp mutations were more frequent in males. The CAL27 cell line carried the TERTp C228T mutation and TERT mRNA expression was 11-15 folds higher compared to non-mutated oral carcinoma cell lines. Conclusions:TERTp C228T and C250T are mutually exclusive and occur at a high frequency in oral rinses and tumor tissues of OSCC patients, showing high concordance between paired samples. These findings support the potential of TERTp mutations as non-invasive biomarkers for OSCC detection. Moreover, their higher prevalence in males suggests possible sex-related differences in OSCC mutation patterns.

Background We have recently shown extensive sequence and conformational homology between tumor-associated antigens (TAAs) and antigens derived from microorganisms (MoAs). The present study aimed to assess the breadth of T-cell recognition specific to MoAs and the corresponding TAAs in healthy subjects (HS) and patients with cancer (CP). Method A library of > 100 peptide-MHC (pMHC) combinations was used to generate DNA-barcode labelled multimers. Homologous peptides were selected from the Cancer Antigenic Peptide Database, as well as Bacteroidetes/Firmicutes-derived peptides. They were incubated with CD8 + T cells from the peripheral blood of HLA-A*02:01 healthy individuals ( n = 10) and cancer patients ( n = 16). T cell recognition was identified using tetramer-staining analysis. Cytotoxicity assay was performed using as target cells TAP-deficient T2 cells loaded with MoA or the paired TuA. Results A total of 66 unique pMHC recognized by CD8+ T cells across all groups were identified. Of these, 21 epitopes from microbiota were identified as novel immunological targets. Reactivity against selected TAAs was observed for both HS and CP. pMHC tetramer staining confirmed CD8+ T cell populations cross-reacting with CTA SSX2 and paired microbiota epitopes. Moreover, PBMCs activated with the MoA where shown to release IFNγ as well as to exert cytotoxic activity against cells presenting the paired TuA. Conclusions Several predicted microbiota-derived MoAs are recognized by T cells in HS and CP. Reactivity against TAAs was observed also in HS, primed by the homologous bacterial antigens. CD8+ T cells cross-reacting with MAGE-A1 and paired microbiota epitopes were identified in three subjects. Therefore, the microbiota can elicit an extensive repertoire of natural memory T cells to TAAs, possibly able to control tumor growth (“natural anti-cancer vaccination”). In addition, non-self MoAs can be included in preventive/therapeutic off-the-shelf cancer vaccines with more potent anti-tumor efficacy than those based on TAAs.

The tumour suppressor factor p53 plays an essential role in regulating numerous cellular processes, including the cell cycle, DNA repair, apoptosis, autophagy, cell metabolism and immune response. TP53 is the most commonly mutated gene in human cancers. These mutations are primarily non‑synonymous changes that produce mutant p53 proteins characterized by loss of function, a dominant negative effect on p53 tetramerisation and gain of function (GOF). GOF mutations not only disrupt the tumour‑suppressive activities of p53 but also endow the mutant proteins with new oncogenic properties. Recent studies analysing different pathogenic features of mutant p53 in cancer‑derived cell lines have demonstrated that restoring wild‑type p53, rather than removing GOF mutations, reduces cancer cell growth. These findings suggest that therapeutic strategies for reactivating wild‑type p53 function in cancer cells may bring a greater benefit than approaches halting mutant p53. This approach could involve the use of small molecules, gene therapy and other methods to re‑establish wild‑type p53 activity. This review describes the complexity of the biological activities of different p53 mutants and summarizes the current therapeutic approaches to restore p53 function.

Growing scientific evidence suggests a link between the expression of toll-like receptor 4 (TLR4) and cervical cancer carcinogenesis. Specifically, a close relation between TLR4 expression and FIGO stage, lymph node metastases, and tumor size has been reported in cervical cancer. In the present study, we aimed to evaluate the relationship between TLR4 expression levels and human papillomavirus (HPV) infection and/or high-risk (hr) HPV integration status in patients with a histological diagnosis of high-grade squamous intraepithelial lesion (H-SIL), and squamous cell carcinoma (SCC) of the uterine cervix. Sixty biopsies of cervical neoplasia, comprising H-SIL (n = 20) and SCC (n = 40), were evaluated for TLR4 expression by immunohistochemistry. All samples were positive for high-risk HPV as confirmed by in situ hybridization (ISH) and broad-spectrum PCR followed by Sanger sequencing analysis. The intensity of TLR4 staining was higher in tissues negative for intraepithelial lesion or malignancy (NILM) than in H-SIL, and further reduced in SCC. Moreover, statistically significant differences have been observed in the percentage of TLR4 expression between NILM and H-SIL and between H-SIL and SCC, with higher percentages of expression in H-SIL than in SCC. Our results showed a significant downregulation of TLR4 in HPV-related H-SIL and SCC, compared to NILM. These data support the hypothesis that TLR4 expression is suppressed in HPV-driven oncogenesis.

Non-coding RNAs have long been recognized for their regulatory roles in various cellular processes, including cancer development and progression. Recent advancements have shed light on a novel aspect of non-coding RNA biology, revealing their ability to encode endogenous peptides also named micropeptides or microprotein through short open reading frames (sORFs). These small proteins play crucial roles in oncogenic processes, acting as either tumour suppressors or tumour promoters, and hold enormous potential as biomarkers for early diagnosis of cancer and as therapeutic targets. This comprehensive review highlights the state of the art on peptides encoded by long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), elucidating their regulatory functions and implications in different cancer types, including breast cancer, hepatocellular carcinoma and colorectal cancer. The review also discusses challenges and future directions in the exploration of these emerging players in cancer biology, emphasizing the importance of further investigation for their clinical translation in diagnosis and therapy.

BACKGROUND:The development of cancer immunotherapeutic strategies relies on the identification and validation of optimal target tumor antigens, which should be tumor-specific as well as able to elicit a swift and potent anti-tumor immune response. The vast majority of such strategies are based on tumor associated antigens (TAAs) which are shared wild type cellular self-epitopes highly expressed on tumor cells. Indeed, TAAs can be used to develop off-the-shelf cancer vaccines appropriate to all patients affected by the same malignancy. However, given that they may be also presented by HLAs on the surface of non-malignant cells, they may be possibly affected by immunological tolerance or elicit autoimmune responses.MAIN BODY:In order to overcome such limitations, analogue peptides with improved antigenicity and immunogenicity able to elicit a cross-reactive T cell response are needed. To this aim, non-self-antigens derived from microorganisms (MoAs) may be of great benefit.

Cancer prevention is one of the aim with the highest priority in order to reduce the burden of cancer diagnosis and treatment on individuals as well as on healthcare systems. To this aim, vaccines represent the most efficient primary cancer prevention strategy. Indeed, anti-cancer immunological memory elicited by preventive vaccines might promptly expand and prevent tumor from progressing. Antigens derived from microorganisms (MoAs), represent the obvious target for developing highly effective preventive vaccines for virus-induced cancers. In this respect, the drastic reduction in cancer incidence following HBV and HPV preventive vaccines are the paradigmatic example of such evidence. More recently, experimental evidences suggest that MoAs may represent a "natural" anti-cancer preventive vaccination or can be exploited for developing vaccines to prevent cancers presenting highly homologous tumor-associated antigens (TAAs) (e.g. molecular mimicry). The present review describes the different preventive anti-cancer vaccines based on antigens derived from pathogens at the different stages of development.

Telomerase activity and telomere elongation are essential conditions for the unlimited proliferation of neoplastic cells. Point mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been found to occur at high frequencies in several tumour types and considered a primary cause of telomerase reactivation in cancer cells. These mutations promote TERT gene expression by multiple mechanisms, including the generation of novel binding sites for nuclear transcription factors, displacement of negative regulators from DNA G-quadruplexes, recruitment of epigenetic activators and disruption of long-range interactions between TERT locus and telomeres. Furthermore, TERT promoter mutations cooperate with TPP1 promoter nucleotide changes to lengthen telomeres and with mutated BRAF and FGFR3 oncoproteins to enhance oncogenic signalling in cancer cells. TERT promoter mutations have been recognized as an early marker of tumour development or a major indicator of poor outcome and reduced patients survival in several cancer types. In this review, we summarize recent findings on the role of TERT promoter mutations, telomerase expression and telomeres elongation in cancer development, their clinical significance and therapeutic opportunities.